Cyclin dependent kinase binding compounds

ABSTRACT

The present invention identifies substances having the property of binding to cyclin dependent kinase (cdk) comprising: (i) a peptide including amino acid residues 84 to 103 of full length p16 protein, or an active portion or derivative thereof; or (ii) a functional mimetic of the fragment. active portion or derivative; the substance excludes full length p16. p15. p18 and p19 proteins. These substances are useful in tumour suppression by inhibiting the phosphorylation of Rb protein. Also described herein is the resolution of the amino acid motifs responsible for binding cdks, an FLD motif. corresponding to amino acid residues 90 to 92 of full length p16 protein. and an LVVL motif, corresponding to amino acid residues 94 to 97 of full length p16 protein. The substances disclosed herein can be used in the treatment of hyperproliferative disorders and to screen and design molecules having the similar properties.

FIELD OF THE INVENTION

[0001] The present invention relates to substances having the propertyof binding to cyclin dependent kinase (cdk), and in particular tosubstances having this property derived from analysis of fragments ofp16 protein. The present invention also relates to pharmaceuticalcompositions comprising these substances and their use in methods ofmedical treatment, especially in the treatment of hyperproliferativedisorders. The invention also relates to methods and uses of thesesubstances in identifying compounds having related activities.

BACKGROUND TO THE INVENTION

[0002] Phosphorylation of the Rb gene product (pRb) by members of thecyclin dependent kinase (cdk) family is an important step in the cellscommitment to undergo mitosis This step is regulated in the later partof the G1 phase of the cell cycle at what is known as the restriction(R) point (6) The cdks are key regulatory factors through which bothpositively and negatively acting cell signal transduction factors merge.Mitogenic stimulation induces an active complex between the D-cyclinsand cdk4 or cdk6 that is capable of phosphorylating pRb in late G1.These kinases are also the targets for cell growth inhibitory signalsarising from contact inhibition, growth factor starvation or TGF-β. Theinhibitory signals can block kinase activity by inducing the productionor activity of different members of the two rapidly enlarging familiesof INK4 and p21/KIP cdk-inhibitors that either interfere directly withthe kinases or with the cyclin-kinase complexes (7). The family of INKproteins that have been identified consists of p15, p16, p18 and p19(20, 22, 23).

[0003] However, unlike p21^(Cip1/wAF1), which is indirectly linked totumour suppression activity through p53 transcriptional stimulation (8),the INK4p16 gene is itself deleted or mutated in a large number of humantumours (9-15). Germ line mutations in INK4p16 have been associated withan increased risk of developing melanoma (9, 10) The 156 amino acidproduct of the INK4p16 gene is known as CDKN2 or p16INK4a (referred toin this application as “p16”)

SUMMARY OF THE INVENTION

[0004] We set out to identify and study the region of p16 that interactswith cyclin dependent kinases such as cdk4 and cdk6, and to investigateapplications of these properties, in particular the possibility that thebinding of cdks by substances comprising a peptide based on this regionof p16 could be used in tumour suppression by inhibiting thephosphorylation of Rb protein.

[0005] Small peptides can sometimes be powerful tools to identifyregions of proteins involved in protein-protein interactions andbiological activity (16-19). In this work, we synthesised a series ofoverlapping 20 amino acid (aa) peptides that spanned the p16 amino acidsequence, and tested the capacity of each biotinylated peptide tointeract with ³⁵S-labelled cdk4 and cdk6 expressed in rabbitreticulocyte lysates

[0006] These experiments identified a 20 amino acid synthetic peptidecorresponding to residues 84 to 103 of p16 that interacts with cdk4 andcdk6, and inhibits cdk4-cyclin D1 mediated phosphorylation of Rb proteinin vitro. An alanine substitution series defined amino acid residuesimportant for the, cdk4 and cdk6 interaction and for the inhibition ofRb phosphorylation. In this application, residues 84 to 103 of p16correspond to the sequence set out in FIG. 1C, i.e.DAAREGFLDTLVVLHRAGAR.

[0007] Further, when coupled to a small peptide carrier molecule andapplied directly to tissue culture medium, the p16-derived peptideblocked cell cycle entrance into S-phase in both serum starved humanHaCaT cells and other types of cells that are cycling normally. This wasassociated with an inhibition of pRb phosphorylation in vivo. Theseresults demonstrate that a p16-derived synthetic peptide coupled to asmall carrier molecule can mimic the G1-phase arrest associated withoverexpression of full length p16 protein. This provides a route to therestoration of the p16 suppressor gene function in human tumours.

[0008] Accordingly, in a first aspect, the present invention provides asubstance having the property of binding to cyclin dependent kinase(cdk) comprising:

[0009] (i) a peptide including amino acid residues 84 to 103 of fulllength p16 protein, or an active portion or derivative thereof; or,

[0010] (ii) a functional mimetic of the fragment, active portion orderivative;

[0011] wherein the substance excludes full length p16, p15, p18 and p1 9proteins;

[0012] Preferably, the cyclin dependent kinase (cdk) is cdk4 or cdk6.The substance preferably also has the property of inhibiting thephosphorylation of Rb protein which is mediated by a complex formedbetween. cdks-and cyclin D. This in turn can be used to block cellulardifferentiation by preventing the entry of cells into the S-phase. Asthe substances bind cyclin dependent kinases, they can also be used toprevent the formation of the complex between cdks and cyclin D, havingthe additional biological effect of increasing cyclin D levels in cells.As well as blocking cdk4 and cdk6 dependent phosphorylation of pRb, thesubstances described herein could be used to target other cellularsubstrates, including the pRb family members p107 and p130, or othersubstances that are targets for cdk4 and cdk6 mediated regulation.

[0013] In the present invention, “an active portion” means a portion ofthe peptide which is less than the full amino acid sequence of thefragment above, but which retains the property of binding to a cyclindependent kinase (cdk) . Preferably, the peptide also has the propertyof inhibiting pRb phosphorylation.

[0014] In the present invention, a “derivative” is a protein modified byvarying the amino acid sequence of the protein, e.g. by manipulation ofthe nucleic acid encoding the protein or by altering the protein itself.Such derivatives of the natural amino acid sequence may involveinsertion, addition, deletion or substitution of one or more aminoacids, without fundamentally altering the essential activity of theproteins. As an example, a derivative of peptide 6 in which asparticacid 92 was substituted for alanine was found to be more potent than thepeptide 6 (residues 84 to 103) in binding to cdk4 and cdk6, and having agreater inhibition of pRb phosphorylation. Other derivatives includeinserting one or more amino acid residues between amino acid motifs FLDand LVVL.

[0015] In the present invention, “functional mimetic” means a substancewhich may not contain a fragment or active portion of, the p16 aminoacid sequence, and probably is not a peptide at all, but which has someor all of the properties of the p16 fragment, in particular the propertyof binding to a cyclin dependent kinase and/or inhibiting pRbphosphorylation.

[0016] In a preferred embodiment, the peptide includes residues 89 to 97of full length p16 protein. More preferably, the peptide includes thepeptide motif FLD, corresponding to amino acids 90 to 92 of full lengthp16 protein, and/or the peptide motif LVVL, corresponding to amino acids94 to 97 of full length p16 protein. We have also found that both the Dand L isoforms of the peptides share the property of binding to cdkand/or inhibiting pRb phosphorylation.

[0017] In a further aspect, the present invention provides compoundscomprising any of the above substances coupled to carrier molecules,enabling the compounds to be delivered to cells in vivo. In oneembodiment, the carrier molecule is a 16 aa peptide sequence derivedfrom the homeodomain of Antennapedia (e.g. as sold under the name“Penetratin”) which can be coupled to one of the above substances via aterminal Cys residue. The “Penetratin” molecule and its properties aredescribed in WO 91/18981.

[0018] In a further aspect, a substance comprising one of the abovepeptides can be stabilised by coupling to another peptide sequence.Preferably, this allows the peptide to adopt a conformation more closelyresembling that of full length p16, typically having the advantage ofincreasing the activity of the peptide relative to the uncoupledfragment, e.g. so that the peptide fragment has an activity more closelyapproaching or surpassing that of full length p16.

[0019] In further aspects, the present invention provides pharmaceuticalcompositions comprising one or more of the above substances and the useof these compositions in methods of medical treatment. In a preferredembodiment, the present invention relates to the use of these substancesin the preparation of medicaments for the treatment ofhyperproliferative disorders, such as cancer, psoriasis orarteriogenesis. In particular, cancers which are p16 negative orassociated with the overexpression of cdks are especially likely torespond well to compositions comprising one or more of the abovesubstances.

[0020] Pharmaceutical compositions according to the present invention,and for use in accordance with the present invention, may comprise, inaddition to one of the above substances, a pharmaceutically acceptableexcipient, carrier, buffer, stabiliser or other materials well known tothose skilled in the art. Such materials should be non-toxic and shouldnot interfere with the efficacy of the active ingredient. The precisenature of the carrier or other material may depend on the route ofadministration, e.g. oral, intravenous, cutaneous or subcutaneous,nasal, intramuscular, intraperitoneal routes. For such, administration,a parenterally acceptable aqueous solution may be employed which ispyrogen-free and has suitable pH, isotonicity and stability. Thoseskilled in the art are well able to prepare suitable solutions.Preservatives, stabilisers, buffers, antioxidants and/or other additivesmay be included, as required. Dosage levels can be determined by thethose skilled in the art, taking into account the disorder to betreated, the condition of the individual patient, the site of delivery,the method of administration and other factors known to practitionersExamples of the techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed) 1980.

[0021] In embodiments in which the substances are proteins, the presentinvention also provides nucleic acid encoding these proteins. Thoseskilled in the art can readily construct such nucleic acid sequencesfrom the amino acid sequences disclosed herein, taking account offactors such as codon preference in the host used to express the nucleicacid sequences. In embodiments of the invention in which the protein iscoupled to a carrier protein, nucleic acid encoding the carrier proteincan be linked to the sequence encoding the. peptides and the sequencesexpressed as a fusion.

[0022] In further aspects, the present invention provides vectorsincorporating the above nucleic acid and host cells transformed with thevectors.

[0023] In a further aspect, the present invention provides the use ofany one of the above substances in screening for (i) compounds havingone or more of the biological activities of the substances describedabove or (ii) compounds which are binding partners of one of thesubstances, e.g. antibodies or complementary peptides specific for p16or a p16 mimetic. Preferably, the substances are peptide fragments ofp16 protein. Examples of screening procedures for mimetics or bindingpartners include:

[0024] (a) immobilising the p16 fragments on a solid support andexposing the support to a library of labelled peptides or othercandidate compounds, and detecting the binding of the peptides orcandidate compound to the p16 fragments;

[0025] (b) using labelled cdks and a library of unlabelled candidatecompound or peptides;

[0026] (c) other combinations of solid phases substrates and bindingmeasurements;

[0027] (d) Western blots using the fragments of p16 protein andantibodies raised to the p16 fragments and determining the displacementof the antibodies by candidate compounds;

[0028] (e) using yeast two hybrid screens to detect candidate peptideswhich bind to the p16 peptide or to oligonucleotides derived from thep16 fragments (for a description of yeast two hybrid screens see ourearlier application WO96/14334);

[0029] (f) using the fragments of p16 protein and/or candidate compoundsin cell systems to determine whether the fragments or candidatecompounds inhibit phosphorylation of Rb and/or prevent the cells fromcycling;

[0030] (g) using the fragments- of p16 protein and/or candidatecompounds in animal models of tumour growth to determine whether thefragments -or candidate compounds prevent the occurrence of tumours,reduce tumour size, inhibit tumour growth and/or inhibit tumour cellmigration.

[0031] In a further aspect, the present invention provides method ofidentifying compounds which compete with one of the above substances,the method comprising:

[0032] (a) binding a predetermined quantity of the substance which isdetectably labelled to a cyclin dependent kinase (cdk);

[0033] (b) adding a candidate compound; and,

[0034] (c) determining the amount of the labelled compound that remainsbound to the cdk or which becomes displaced by the candidate compound

[0035] In a further aspect, the present invention provides a method ofidentifying mimetics of one of the above substances, the methodcomprising:

[0036] (a) immobilising one or more candidate compounds on a solidsubstrate;

[0037] (b) exposing the substrate to a labelled cyclin dependent kinase(cdk);

[0038] (c) selecting the candidate compounds that bind to cdk.

[0039] In the above aspects, preferably the cyclin dependent kinase iscdk4 or cdk6. Preferably, the substance is a fragment of p16 protein,and more preferably the FLD and LVVL motifs disclosed above.Conveniently, the candidate compounds can be selected from a syntheticcombinatorial library.

[0040] The present invention may further comprise testing the candidatecompound for the property of inhibiting pRb phosphorylation and/ortesting the compound for the property of inhibiting the entry of cellsinto the S-phase.

[0041] In a further aspect, the present invention provides the use of afragment of p16 protein including the amino acid motifs FLD,corresponding to amino acid residues 90 to 92 of full length p16protein, and/or LVVL, corresponding to amino acid residues 94 to 97 offull length p16 protein in the design of an organic compound which ismodelled to resemble the three dimensional structure of said amino acidmotifs, the organic compound having the properties of binding to cyclindependent kinase and/or inhibiting pRb phosphorylation.

[0042] The designing of mimetics to a known pharmaceutically activecompound is a known approach to the development of pharmaceuticals basedon a “lead” compound. This might be desirable where the active compoundis difficult or expensive to synthesise or where it is unsuitable for aparticular method of administration, eg peptides are unsuitable activeagents for oral compositions as they tend to be quickly degraded byproteases in the alimentary canal. Mimetic design, synthesis and testingis generally used to avoid randomly screening large number of moleculesfor a target property.

[0043] There are several steps commonly taken in the design of a mimeticfrom a compound having a given target property. Firstly, the particularparts of the compound that are critical and/or important in determiningthe target property are determined. In the -case of a peptide, this canbe done by systematically varying the amino acid residues in thepeptide, eg by substituting each residue in turn. These parts orresidues constituting the active region of the compound are known as its“pharmacophore”.

[0044] Once the pharmacophore has been found, its structure is modelledto according its physical properties, eg stereochemistry, bonding, sizeand/or charge, using data from a range of sources, eg spectroscopictechniques, X-ray diffraction data and NMR. Computational analysis,similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modelling process.

[0045] In a variant of this approach, the three-dimensional structure ofthe ligand and its binding partner are modelled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this in the design of themimetic.

[0046] A template molecule is then selected onto which chemical groupswhich mimic the pharmacophore can be grafted. The template molecule andthe chemical groups grafted on to it can conveniently be selected sothat the mimetic is easy to synthesise, is likely to bepharmacologically acceptable, and does not degrade in vivo, whileretaining the biological activity of the lead compound. The mimetic ormimetics found by this approach can then be screened to see whether theyhave the target property, or to what extent they exhibit it. Furtheroptimisation or modification can then be carried out to arrive at one ormore final mimetics for in vivo or clinical testing.

[0047] By way of example, the present invention will now be described inmore detail with reference to the accompanying figures. The followingexamples are provided to illustrate the present invention, and shouldnot be interpreted as limiting the scope of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048]FIGS. 1A and B show the relative binding of the p16 derivedpeptides to in vitro expressed cdk4 or cdk6.

[0049]FIG. 1C shows peptide 6 corresponds to amino acids 84 to 103 ofthe p16 protein.

[0050]FIGS. 1D and E show similar binding to in vitro translated cdk4and cdk6 of an alanine substitution series of peptide 6 amino acids. Theamino acid residues substituted by alanine are indicated and therelative amount of cdk4 and cdk6 precipitated by each peptide is shown.Substituting the hydrophobic residues corresponding to amino acids 89-90and 94-97 to alanine decreases the binding of peptide 6 to both kinases,while substitution of Asp92 significantly increases the interaction.

[0051]FIG. 1F shows results from an experiment in which Sf9 insect celllysates containing cdk4 were incubated with the following biotinylatedpeptides: peptide 6 (lanes 1 and 4), peptide 1 (lanes 2 and 5) andpeptide 10 (lanes 3 and 8). The complexes were precipitated withstreptavidin-coated agarose beads. The lower of the cdk4 bands isrelated to the extraction method (see Materials and Methods). Theseresults show that if cyclin Di containing insect cell lysates were addedto cdk4 prior to the addition of peptide, the extracts containing thepeptide can not bind the cdk4. However, if the peptides are added to thecdk4 prior to cyclin D1, the peptide binds cdk4, suggesting that thepeptides can be sued to interfere in the binding between cyclin D andcdks.

[0052]FIG. 2A shows the inhibition of phosphorylation of E. coliexpressed and purified full length Rb protein. p16 derived wild typepeptides (peptides 1, 6 and 10) or the peptide 6 alanine substitutionseries were tested for their capacity to interfere with pRbphosphorylation by lysates from Sf9 insect cells containing cdk4.Peptides 1 and 10 of the p16 series do not precipitate cdk4 or cdk6 anddo not affect pRb phosphorylation. Peptide 6 binds to cdk4 and cdk6 andsignificantly reduces the level of pRb phosphorylation. The amino acidresidues of the peptide 6 substituted with alanine are indicated and thelevels of pRb phosphorylation in the presence of each peptide are shown.The Ala-Asp substitution at residue 92 (Ala92) inhibits phosphorylationof the full length Rb protein even more efficiently than the wild typepeptide 6, reflecting its more potent binding to cdk4 and cdk6. Alaninesubstitution of the hydrophobic residues at positions 90 and 95-97 orclosely adjacent residues reduced the inhibitory capacity to backgroundlevels.

[0053]FIG. 2B shows the effect of increasing amounts of peptides 6,Ala92 and Ala94 on pRb phosphorylation. At 50 μM the block ofcdk4-cyclin D dependent pRb phosphorylation by peptide Ala-92 is nearlycomplete.

[0054]FIG. 3 shows the inhibition of S-phase entry, of humankeratinocyte derived HaCaT cells with peptide 6 coupled to a smallpeptide carrier. Cells were synchronised in G0 by serum starvation for72 hours before serum and 10 μM BrdU were added. FIG. 3A shows theindicated time points after serum stimulation when 100 nM peptide 6coupled to the Penetratin carrier molecule was added to the tissueculture medium. The data presented show % inhibition of cells enteringS-phase after incubation with peptide 6 coupled to Penetratin inrelation to cells incubated with serum only

[0055]FIG. 3B sets out panels A, C, E and G which show cellssynthesising DNA by BrdU labelling, and panels B, D, F and H show thesame fields of cells stained with Hoescht. The percentage of cells thatincorporated BrdU after serum stimulation was 71% at 24 hours (E and F)and 14% (G and H) at 3 hours. The number of cells incorporating BrdU at24 hours was significantly reduced when peptide 6 coupled to thePenetratin carrier molecule was added at 12 hours (panel A and B)compared to at 14 hours (panel C and D). No effect on DNA synthesiscould be observed with Penetratin only (not shown).

[0056]FIG. 4A shows the phosphorylation of pRb in vivo.Hyperphosphorylated pRb has a lower affinity for the nuclear compartmentcompared to the hypophosphorylated subtypes and can be extracted fromthe nucleus by using a hypotonic buffer containing Triton X-100(26).Panels A, C and E show staining with anti-pRb monoclonal antibody and B,D and F show the same field of cells stained with Hoescht. HaCaT cellswere serum starved for 72 hours before serum addition. Peptide 6 coupledto Penetratin (panel C) or Penetratin by itself (panel A) was added at 8hours after serum addition at 100 nM and the amount of phosphorylatedpRb was estimated at 23 hours by determining pRb extractability (panelsA and C) compared with a non-extracted staining (panel E).

[0057]FIG. 4B shows the status of pRb phosphorylation in HaCaT wholecell extracts as determined by Western blot analysis. Cells were starvedfor 72 hours before serum was added and harvested at the indicated timepoints. Peptide 6 coupled to Penetratin or Penetratin by itself wasadded at 10 hours as indicated.

[0058]FIG. 5A shows the relative inhibition of pRb phosphorylation byp16, related peptides from corresponding regions of other INK familymembers and fragments of p16. In particular, it shows the effect ofreducing the size of the p16 fragment on pRb phosphorylation. FIGS. 5Band 5C show the corresponding determination of the binding of the INKfamily members and fragments to cdk4 and cdk6.

[0059]FIGS. 6A and 6B show the graphs indicating the effect ofincreasing concentrations of peptides 20 and 21 (FIG. 6A) and wt p16 andV95,96A p16 (FIG. 6B) on the inhibition of cdk-cyclin D kinase activity.Peptide 20 is a 36 aa long synthetic peptide that carries the D92Amutation and peptide 21 carries the VV95,96AA mutation.

[0060]FIG. 7 shows the results from FACS analysis of HaCaT cells treatedwith three different peptides 36 aa peptide consisting of a 16 aaPenetratin sequence coupled to a 20 aa peptide (peptide 6, the V95,96Aand the D92A mutant p16 peptide) so that the Penetratin sequence is atthe N-terminus of the 20 aa peptides (i.e. N-Penetratin-p16-C). In theseexperiments, the cells were starved for 72 hours before 10% FCS and thepeptides were added and analysed 2 hours later.

DETAILED DESCRIPTION Materials and Methods

[0061] Peptide Precipitation

[0062] A 20 aa peptide library with a 5 aa overlap of p16 (apart fromthe first 8 N-terminal residues) was synthesised adding a SGSG linker tothe N-terminus to which a biotin group was coupled. The alaninesubstitution series of peptide 6 was synthesised in the same way. Thepeptides were coupled to streptavidin immobilised on agarose beads andwashed 4 times in PBS before incubating for 1 hour on ice with rabbitreticulocyte lysate (Promega) containing ³⁵S-methionine labelled cdk4or-cdk6. The beads were washed 4 times in 1.2×PBS with 0.2% Triton X-100before addition of SDS loading buffer and applied to 12% SDSpolyacrylamide gels. The gels were exposed to an autoradiography filmand the bands corresponding to cdk4 and cdk6 were analysed bydensitometry.

[0063] pRb Phosphorylation in vitro

[0064] Peptides were incubated at a concentration of 25 mM in a buffercontaining 50 mM Hepes pH 7.4, 10 mM MgCl₂, 2.5 mM EGTA, 1 mM DTT, 10 mMβ-glycerophosphate, 1 mM NaF and 1 mM Na₃V0₄ and 3 ml of extract fromSf9 insect cells infected with human cdk4-expressing baculovirus lysedin 10 mM Hepes pH 7.4, 10 mM NaCl, 1 mM EDTA and 0.5 mM PMSF. Themixture was incubated for 60 minutes on ice. Human cyclin D containingSf9 lysate (3 ml) prepared as above was added together with 0.6 mg ofpurified recombinant full length Rb protein and 2.5 m ³²P ATP in a finalconcentration of 50 mM ATP and incubated for 10 minutes at +30° C., thereaction was terminated by addition of SDS loading buffer and loadedonto 8%; SDS polyacrylamide gels. The gels were either exposed toautoradiographic film (FIG. 2A) or the levels of pRb phosphorylationwere estimated by a Phosphoimager (FIG. 2B).

[0065] Cell Cycle Inhibition

[0066] A cysteine residue was added to the C-terminus of peptide 6 andused for coupling to the 16 amino acids long Penetratin peptide (aminoacid sequence RQIKIWFQNRRMKWKK) of the Antennapedia homeodomain (24)(Appligen) by means of a disulphide bond. Cells were seeded on coverslips prior to starvation for 72 hours in DMEM medium without FCS. Themedium was substituted by DMEM containing 10% FCS and BrdU. The coupledpeptides were added at different time points after serum stimulation.The number of cells entering S-phase was determined by estimating thenumbers of cells incorporating BrdU at 24 hours by fixing the cells oncover slips in acetone/methanol (1:1), incubating in 1M HCl for 30minutes, washing 6 times in PBS and then incubating with anti-BrdUmonoclonal antibody and Texas Red conjugated secondary antibody andmounted in Mowiol containing Hoescht. At least six different areas onthree different cover slips were counted for each single experimentwhich was repeated at least two times. The values presented in FIG. 3Ashow one representative experiment.

[0067] FACS Analysis

[0068] Twenty minutes before harvest, cells were incubated with 10 μMBrdU. Tryptinised cells were then washed in PBS and resuspended in 1 mlof PBS and carefully mixed with 3 ml of 96% EtOH and incubated for 1hour at 4° C. The cells were then incubated in 2 ml of 30 mM HClcontaining 1 mg/ml of Pepstatin for 30 mins at 37° C. before incubationin 2M HCl for 15 minutes. After careful washing 6 times in PBS, thecells were incubated in 200 μl (1:50) of anti-BrdU antibodies (BectonDickinson) for 1 hour at room temperature. After washing in PBS, thecells were incubated in FITC conjugated anti-mouse IgG (1:80) (Sigma)for 30 mins. After washing, the cells were resuspended in PBS containing25 μg/ml of Propidium Iodine and analysed on FACS.

[0069] pRb Phosphorylation in vivo

[0070] Hyperphosphorylated pRb was extracted from cells cultured oncover slips by treating the cells with hypotonic buffer containing 0.1%Triton X-100 prior to fixation in acetone/methanol (1:1) (26). Fixedcells were incubated for 1 hour with anti-pRb monoclonal antibody IF8,washed 3 times in PBS and incubated for 45 minutes with TexasRedconjugated secondary antibody before the cover slips were mounted inMowiol containing Hoescht.

[0071] For Western blot analysis, cells were lysed in RIPA buffercontaining 50 mM Tris pH 8.0, 150 mM NaCl, 1.0% NP-40, 0:5% DOC, 0.1%SDS and 0.1 mM PMSF for 30 minutes at +4° C. The protein concentrationswere determined before-the samples were boiled in SDS loading buffer,run on 8% SDS polyacrylamide gels and transferred to a nitrocellulosemembrane. The filters were first incubated with anti-pRb monoclonalantibody IF8 before being incubated with a horse (DAKO) and developedwith ECL (Amersham).

[0072] Site Directed Mutagenesis

[0073] The VV95,96AA mutations were introduced into wild type his-tagged p16 protein using the transformation site-directed mutagenesiskit from Promega, according to manufacturer's instructions. Themutations were introduced by changing the corresponding codons from GTGGTG to GCG GCG and then confirming the sequence by DNA sequencing.

[0074] Results

[0075] Identification of a region of p16 that binds to cdks

[0076]FIGS. 1A and B show that a peptide corresponding to aa 84 to 103of p16 (peptide 6) when coupled to streptavidin agarose beads can beused to extract both cdk4 and cdk6 from the reticulocyte lysates. Analanine substitution series of this peptide (FIG. 1C) revealed thatsubstitution of hydrophobic amino acids in the region between residues89 to 96 decreased the capacity of the peptide to bind both cdk4 andcdk6. Interestingly, substituion of aspartic acid 92 with alaninesignificantly increased the binding of the peptide to both kinases(FIGS. 1D and E).

[0077] p16 Fragments and the Inhibition of pRb Phosphorylation

[0078] In order to study the functional significance of p16 peptideinteractions with the cyclin dependent kinases we asked whether the p16derived peptides, as well as the alanine-substitution series of peptide6, affected cdk4-cyclin D ability to phosphorylate pRb in an in vitroassay (FIG. 2). Only peptide 6 of the p16-derived series significantlydecreased pRb phosphorylation (only results from peptides 1, 6 and 10are shown) A correlation was observed between the capacity of thevarious peptides in the alanine-substitution series to bind cdk4 andcdk6 and the level of inhibition of cdk4-cyclin D1 kinase activity.

[0079] The Effect of Mutations in the p16 Binding Domain

[0080] Most significantly, substitution of amino acids in, or adjacentto, the two hydrophobic regions located between residues correspondingto aa 89 to 96 of the full length p16 protein resulted in a decrease ofthe peptide 6 induced inhibition of pRb phosphorylation. Interestingly,the change of aspartic acid 92 to alanine resulted in a peptide morepotent than peptide 6 in inhibition of the cdk4 kinase activity. Adilution series revealed that at 50 AM peptide concentration, pRbphosphorylation was almost completely blocked by the Ala92 peptide andpeptide 6. In contrast, the single substitution Ala94 completelyinactivates the function of peptide 6 in this assay (FIG. 2B). Thecorrelation between the enhanced binding of the Ala92 peptide and itsgreater efficiency as a kinase inhibitor is provocative and suggeststhat further variants of the peptide 6 sequence might possess greateractivity, perhaps by encouraging the peptide to adopt a conformationmore similar to that of the active inhibitory site on the native p16protein. A comparison of the work described here in identifying theinhibitory region of p16 represented by peptide 6 and other relatedproteins shows that an identical motif is present in the correspondingdomain of the kinase inhibitor p15 (20,21) and is conserved in theclosely related p18 (21) and, p19 (22,23) inhibitors, although prior tothis work this similarity and its significance was not realised by thoseskilled in the art.

[0081] Point mutations in the p16 gene have been found in tumours fromfamilial and primary melanomas as well as in tumours from the oesophagusand the bladder (9, 10, 14, 15). Some encompassed by peptide 6, and havebeen shown to have lost their ability to inhibit cell proliferation andpRb phosphorylation(3,4,12). This further supports the importance ofthis region for p16 protein function.

[0082] Mutations in the p16 protein that result in its inactivation andare outside of the region suggested above that mediates the binding tocdk have been shown to induce global conformation changes of the p16protein or are temperature sensitive, suggesting that these mutationsmight affect the structure of the domain we suggest mediates binding.Furthermore, deletion of the p16 N- or C-terminus both result in p16inactivation, supporting the deduction that the structure of p16 issensitive.

Effect of p16 on Cell Proliferation and the Use of Carrier Peptides

[0083] Since overexpression of p16 in cultured cells can block S-phaseentry (1-4), we wanted to see if peptide 6 could affect cellproliferation. A 16 aa region of the Antennapedia homeodomain that hasbeen shown to translocate through biological membranes in a rapid andenergy independent fashion (24) was coupled as carrier to peptide 6 andadded to the tissue culture medium of serum-starved human keratinocytederived HaCaT cells.

[0084]FIG. 3 shows that when 0.1 μM of the p16 peptide coupled to thecarrier molecule was added at the same time or up to 12 hours after theaddition of serum, the number of cells entering S-phase were reduceddramatically according to BrdU incorporation measured at 24 hours afterserum addition. However, when the coupled peptide was added to themedium 14 hours after addition of serum, the number of cells enteringS-phase was the same as that seen in cells not treated with peptide.This suggests that the effect of the peptide is limited to a rathernarrow window in the cell cycle that corresponds to the later part ofG1. This includes the restriction (R) point at which serum stimulationand protein synthesis are no longer required to ensure entry intoS-phase and which has been suggested to be the critical time of pRbphosphorylation (6,25).

[0085] When starved cells were incubated with the p16 peptide coupled tothe carrier molecule or the carrier molecule alone at 10 hours postserum addition a difference in pRb extractability could be observed whenassayed at 23 hours(26). Approximately 60%; of the cells incubated withpeptide 6 stained with an anti-pRb monoclonal antibody compared to only14% incubated with the carrier only (FIG. 4A). This observation wasconfirmed by Western blot analysis of whole HaCaT cell extracts treatedin a similar fashion (FIG. 4B). The results suggest that the number ofcells carrying hypophosphorylated pRb increased significantly at 23hours after serum stimulation when peptide 6 coupled to the carrierpeptide was added before 12 hours. It also implies that the inhibitionof cdk4-cyclin D activity observed in baculovirus infected Sf9 cellextracts (FIG. 2) is taking place in vivo.

[0086] The consistent inhibition of S-phase entry after adding thecoupled peptides between 0 and 12 hours suggests that the effect of thepeptide is persistent and that the carrier linked peptide is not rapidlydegraded in the cells. This is consistent with reports suggesting thatthe Antennapedia homeodomain carrier peptide is protected fromproteolytic degradation in the cell (24).

[0087] Refinement of the cdk Binding Motifs of p16 and Comparison withother Proteins

[0088] The results above show that a 20 aa peptide derived from thethird ankyrin like repeat of p16 has similar features as the full lengthprotein, e.g. binding to the cyclin-cell dependent kinases cdk4 and cdk6and to inhibit cdk4-cyclin D1 kinase activity in vitro as well as toblock cell cycle progression. This region included the peptide sequencethat corresponds to aa 84 to 103 of the full length p16 protein and isidentical to the corresponding region of p15 and highly conserved in p18and p19 as well as in the mouse p16.

[0089] Since members of the INK family of kinase inhibitors inhibitCDK-cyclin D kinase activity specifically by direct interaction withcdk4-and cdk6, we wanted to see whether this activity can be determinedto one highly conserved domain shared between these proteins. Since itis mainly p16, and to a lesser extent p15, of the INK family that isassociated with tumour suppressor activity it will be important to knowif these different proteins inhibit the CDK-cyclin D kinase complex in asimilar fashion through the same domain suggesting that the regulationof expression of these different proteins determines their role astumour suppressors rather than their mean of action. Thus, we carriedout experiments to see if this peptide domain could be further minimizedand substituted with modified amino acid residues that are insensitiveto protease degradation in order to improve its potential as a model fora synthetic tumour suppressor peptide.

[0090] To study the binding of peptides to cdk4 and cdk6 we expressedthe proteins in a coupled in vitro reticulocyte translation system inthe presence of ³⁵S labelled methionine. A biotin group coupled to aSer-Gly-Ser-Gly-linker at the N-terminus of the peptides was coupled tostreptavidin coated agarose beads and incubated with the cell lysates.FIG. 5A shows the results measuring pRb phosphorylation, with FIGS. 5Band 5C showing the determination of the binding of the INK familymembers and the p16 peptide fragments to cdk4 and cdk6.

[0091] FIGS. 5A-C show that peptides, corresponding to the 84-103 regionof p16, derived from p18 and mouse p16 inhibit pRb phosphorylation, asreflected in their capacity to inhibit pRb phosphorylation by Sf9 insectcell lysates overexpressing cdk4 and cyclin D1, and binds to both cdk4and cdk6 in a similar way.

[0092] We then tested a p16 peptide deletion series which was made bydeleting two residues at the same time from either the N- or C-terminus.We found that removing 2 residues-at the N-terminus (peptide 6 in FIGS.5A-C) severely reduced, both the cdk binding and the kinase inhibitoryeffect and that the activity could be restored when another two residueswere deleted at either terminus. Peptide 10 only includes the 10residues that correspond to a motif that is conserved among ankyrin likerepeats and is predicted to form a tight secondary helical structure.This peptide has lost some of its binding capacity but is still a goodkinase inhibitor demonstrating that the original 20 amino acid. p16peptide can be reduced with at least 10 residues and still inhibitCDK-cyclin D1 kinase activity. This deletion series and the alanine scanshows that in the peptides we examined there was a strong correlationbetween binding and kinase inhibit on.

[0093] It is interesting to notice that the R87P substitution does nothamper the function of the peptide. Since this mutation, like most p16mutations so far detected, is located outside the region that is shown.to be important for the peptide interaction with the cdk, it suggeststhat these mutations will induce conformational changes of the proteinthat will effect the central ankyrin like domain. This hypothesis isstrengthened by recent NMR studies showing that P114 and G101W give riseto global conformational changes of the protein and that the R87Pmutation has been shown to be temperature sensitive. Similarexplanations might also give an answer to the surprising observationsthat the peptide loses its effect when two residues are taken off theN-terminus, suggesting that these deletions causes conformationalchanges of the peptide. The minimal binding peptide (peptide 10)basically consists of two hydrophobic regions surrounded by polarresidues that could form an amphipatic helical wheel. The p1 8 peptidecarries 8 substitutions, compared to the p16 peptide, and the QT atpositions 95 and 96 would at first seem to disturb the binding domain ofthe peptide since these disrupts the second hydrophobic pocket. However,this peptide carries two hydrophobic residues at position 97 and 98instead which are surrounded by polar residues and it also has the GFLDregion intact, as well as leucine 98, which might suggest that thesecond hydrophobic pocket can be moved a few residues toward theC-terminus without effecting cdk binding.

[0094] Thus, these results show that after initial reductions in thesize of the p16 fragments resulted some reduction in cdk inhibition (seepeptide 6), further reduction in the size of the p16 fragments onceagain increased the inhibition- (peptides 7 to 10). This demonstratesthat small peptides comprising 16 aa (peptide 7), 14 aa (peptide 8), 12aa (peptide 9) and 10 aa (peptide 10) are able to exert a biologicaleffect which is the same or analogous to that of full length p16. Theseresults also support the idea that two motifs of p16 are important forkinase binding, a FLD motif and a LVVL motif.

[0095] Accordingly, these results indicate that the 20 aa fragment ofp16 disclosed above (residues 84 to 103) can be made at least 50 smallerand still be active.

[0096] Alanine Substitution at Positions 95 and 96

[0097] The results above from an alanine scan substitution series of thep16 peptide suggested that the two valines at position 95 and 96 couldbe important for the binding of the peptide to cdk and for its kinaseinhibitory function, and that substitution of aspartic acid 92 toalanine would potentiation both binding and kinase inhibitory capacityIt is also clear from the peptide deletion series that these residuesare within the 10 residues that mediate binding of the peptide.

[0098] Accordingly, to investigate this further, we introduced theVV95;96AA and the D92A mutations into the highly purified peptides thatwere linked to the third domain of the Anteinapedia homeodomain fortransporting peptides across biological membranes and the W95;96AA intothe full length is protein in order to see if residues that seemimportant for peptide binding to cdk4 and cdk6 also influenced thebinding off the full length protein. The VV95;96AA mutations wereintroduced into a His-tagged wild type p16 and expressed in E. coli andthe corresponding peptides were synthesized at 99.9% purity.

[0099]FIGS. 6A and 6B show the results of these studies. The resultsshow that peptide 21, which has the two VV95,96AA substitutions, issignificantly less active in vivo. The same peptide is also less activein inhibiting the cdk-cyclin D kinase activity in vitro. This confirmsthe results of the first alanine scan described above which suggestedthat these residues are important for the peptide to bind to cdk4 andcdk6.

[0100] When the same substitutions are put into the wild type His-taggedp16 protein a similar loss of kinase inhibitory activity is observed (inboth cases the 50% kinase inhibitory concentration is increased about5-fold). While we do not wish to be bound by, any particular theory,these results further support the view that these residues are involvedin direct binding of the peptide fragments of p16, as well as the fulllength protein, to cdk4 and cdk6, i.e. the mechanism of kinaseinhibition is the same in the full length p16 and the peptide fragmentsdescribed here.

[0101] In vivo, at the same concentration, the peptide 20 induces analmost complete block of S-phase entry in HaCaT cells, whereas the pep21has only marginal effect at a 10 μM peptide concentration.

[0102] Response to p16 in Different Cell Lines

[0103] Mouse embryonic fibroblast (MEF) from mice that are p16(−/−)(knockouts) and from normal p16(+/+) were tested for response to the p16peptides linked to Penetratin described above. After 12 hours oftreatment with the same amount of the p16 peptide linked to Penetratinthere is an increase of 42i of the (−/−) cells population in G1 comparedto 9% of the (+/+). After 24 hours the figure is 22% compared to 5%. Atthe same time the decrease in S phase is 33% for the (+/+) 30%. Takentogether, these results suggest that p16(−/−) MEFs are more sensitive tothe p16 peptide than the (+/+).

[0104] We have also tested some other cell lines and we see effect incells derived from fibroblast, epithelial and muscle origin, and theseshow a similar suppression of growth in response to p16 peptide. By wayof comparison, no growth suppression was observed in a Saos 2 (pRbnegative cell line), confirming the biological action of p16 is throughthe inhibition of pRb phosphorylation. These results are summarised inTable 1.

[0105] We have also carried out experiments to see whether p16 peptidewill inhibit the differentiation of mouse myoblast cells (C2C12 cells)into myotube cells. These results show that when C2C12 cells are put in0.5% FCS medium they will stop growing and from multinucleated myotubes.However, if they are treated with the p16 peptide they will, in additionto stopping growing, also inhibit the formation of multinucleatedmyotubes in the presence of 0.5% FCS. TABLE 1 Tested Growth suppressorcell lines effect by p16 peptide Source p16(−/−) ++ C2C12 ++ HaCaT +++human keratinocyte line human primary +++ keratinocytes MRC5 + humanfibroblast MCF7 +++ human breast cancer derived line MEF+/+ + mouseembiyo fibroblast p16 positive 3T3 ++ mouse fibroblast line Saos2 − pRbnegative tumour cell line

[0106] We have also observed that epithelial cells treated with the p16peptide will alter the morphology of the cell-colonies to a more denseand rounded up phenotype. This is associated with an increase in thecells adhesion to the tissue culture dish resulting in 6×increase intime before the cells will come off after trypsin treatment,demonstrating that the peptides will change cell adhesion properties.The modification of cellular adhesion properties has applications inpreventing the spread of a tumour to form secondary tumours, providing amethod for modulating the invasive capacity of tumour cells.

[0107] Finally, preliminary results suggest that the peptide areassociated with senescence in human keratinocyte derived cells (HaCaTcells) as determined by a Beta-Gal assays. This is also important sinceit has been suggested that one of the physiological functions of the p16protein involves senescence mechanisms which might be linked to itstumour suppressor role in vivo. The assay for identifying senescence incells is described in Dimri et al, P.N.A.S., 1995, page 9396 seq.

[0108] FACS Analysis

[0109]FIG. 7 shows the results from FACS analysis of HaCaT cells treatedwith three different peptides 36 aa peptide consisting of a 16aaPenetratin sequence coupled to a 20aa peptide (peptide 6, the V95,96Aand the D92A mutant p16 peptide). These graphs show that the cells thatdo not get FCS are in the G1 phase and have been arrested properly bythe addition of the peptides.

[0110] Conclusions

[0111] These results demonstrate that a 20 aa synthetic peptidecorresponding to residues 84 to 103 of the p16 protein can mimicessential biochemical and biological properties described for the fulllength wild-type p16 protein. Most important is the discovery that thepeptide coupled to a small carrier molecule has the capacity to inhibitcell proliferation in vivo after direct addition to the tissue culturemedium. This method generically broadens the application of smallpeptides in studying biological events in vivo and, in this case, mayallow them to be used to replace specific suppressor gene function fortherapeutic applications and to serve as models for identifying targetsfor novel anti-proliferative drugs.

[0112] As a number of different tumours show defects in the pRbphosphorylation regulatory pathway, including over expression of cdk4and cyclin D1, as well as showing loss of p16 function. All thesetumours are potential candidates for a drug that would inhibitcdk-cyclin D activity in vivo.

REFERENCES

[0113] The references mentioned in this application are all hereinincorporated by reference.

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1 16 1 20 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide 1 Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu Val ValLeu His Arg 1 5 10 15 Ala Gly Ala Arg 20 2 4 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide 2 Leu Val Val Leu 13 16 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide 3 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met LysTrp Lys Lys 1 5 10 15 4 4 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide 4 Ser Gly Ser Gly 1 5 4 PRTArtificial Sequence Description of Artificial Sequence synthetic peptide5 Gly Phe Leu Asp 1 6 8 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide 6 Phe Leu Asp Xaa Leu Val Val Leu1 5 7 20 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide 7 Asp Ala Ala Arg Ala Gly Phe Leu Asp Thr Leu Gln ThrLeu Leu Glu 1 5 10 15 Phe Gln Ala Asp 20 8 20 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide 8 Asp Ala Ala ArgGlu Gly Phe Leu Asp Thr Leu Val Val Leu His Gly 1 5 10 15 Ser Gly AlaArg 20 9 20 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide 9 Asp Ala Ala Pro Glu Gly Phe Leu Asp Thr Leu Val ValLeu His Arg 1 5 10 15 Ala Gly Ala Arg 20 10 20 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide 10 Asp Ala Ala ArgGlu Gly Phe Leu Asp Thr Leu Val Val Leu His Arg 1 5 10 15 Ala Gly AlaArg 20 11 16 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide 11 Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu Val ValLeu His Arg 1 5 10 15 12 18 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide 12 Ala Arg Glu Gly Phe Leu Asp ThrLeu Val Val Leu His Arg Ala Gly 1 5 10 15 Ala Arg 13 16 PRT ArtificialSequence Description of Artificial Sequence synthetic peptide 13 Glu GlyPhe Leu Asp Thr Leu Val Val Leu His Arg Ala Gly Ala Arg 1 5 10 15 14 14PRT Artificial Sequence Description of Artificial Sequence syntheticpeptide 14 Phe Leu Asp Thr Leu Val Val Leu His Arg Ala Gly Ala Arg 1 510 15 12 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide 15 Phe Leu Asp Thr Leu Val Val Leu His Arg Ala Gly 1 510 16 10 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide 16 Phe Leu Asp Thr Leu Val Val Leu His Arg 1 5 10

1. A substance having the property of binding to cyclin dependent kinase(cdk) comprising: (i) a peptide including amino acid residues 84 to 103of full length p16 protein, or an active portion or derivative thereof;or, (ii) a functional mimetic of the fragment, active portion orderivative; wherein the substance excludes full length p16, p15, p18 andp19 proteins.
 2. The substance of claim 1 wherein the cyclin dependentkinase is cdk4 or cdk6.
 3. The substance of claim 1 or claim 2 whereinbinding of the substance to the cyclin dependent kinase causesinhibition of pRb phosphorylation.
 4. The substance of any one of claims1 to 3 wherein the peptide includes amino acid residues 89 to 97 of fulllength p16 protein.
 5. The substance of any one of the preceding claimswherein the peptide fragment includes the amino acid motifs FLD,corresponding to amino acid residues 90 to 92 of full length p16protein, and/or LVVL, corresponding to amino acid residues 94 to 97 offull length p16 protein.
 6. The substance of claim 5 wherein the peptidefragment includes the amino acid motif FLDxLVVL, wherein x one or moreamino acids.
 7. The substance of any one of the preceding claims whereinthe aspartic acid residue in the peptide fragment at the positioncorresponding to position 92 of full length p16 protein is substitutedfor a hydrophobic amino acid residue.
 8. The substance of claim 8wherein the hydrophobic amino acid residue is alanine.
 9. The substanceof any one of the preceding claims derived from p16, p15, p18 or p19protein.
 10. The substance of any one of the preceding claims coupled toa carrier molecule so that the substance can be delivered to cells. 11.The substance of claim 10 wherein the carrier molecule comprises apeptide derived from an Ancennapedia homeodomain.
 12. The substance ofany one of the preceding claims wherein the peptide fragment is coupledto a stabilising molecule.
 13. A pharmaceutical composition comprisingone or more of the substances of any one of the preceding claims, incombination with a physiologically acceptable carrier.
 14. A substanceof any one of claims 1 to 12 for use in a method of medical treatment.15. The use of a substance of any one of claims 1 to 12 in thepreparation of a medicament for the treatment of a hyperproliferativedisorder.
 16. The use of claim 15 wherein the hyperproliferativedisorder is cancer, psoriasis or arteriogenesis.
 17. Nucleic acidencoding the substances of any one of claims 1 to
 12. 18. A vectorincorporating the nucleic acid of claim 17 operably linked to expressioncontrol sequences.
 19. The use of a substance of any one of claims 1 to12 in a method of in screening for (i) compounds having the one or moreof the biological activities of the substances described above or (ii)compounds which are binding partners of one of the substances.
 20. Amethod of identifying compounds which compete with a substance of anyone of claims 1 to 12, the method comprising: (a) binding apredetermined quantity of the substance which is detectably labelled toa cyclin dependent kinase (cdk); (b) adding a candidate compound; and,(c) determining the amount of the labelled compound that remains boundto thecdk or which becomes displaced by the candidate compound.
 21. Amethod of identifying mimetics of a substance of any one of claims 1 to12, the method comprising: (a) immobilising one or more candidatecompounds on a solid substrate; (b) exposing the substrate to a labelledcyclin dependent kinase (cdk); (c) selecting the candidate compoundsthat bind to cdk.
 22. The method of claim 20 or claim 21 wherein thecdks are produced in reticulocyte lysates.
 23. The method of any one ofclaims 20 to 22 wherein the cell dependent kinase is cdk4 or cdk6. 24.The method of any one of claims 20 to 23, further comprising testing thecandidate compound for the property of inhibiting pRb phosphorylationand/or testing the compound for the property of inhibiting the entry ofcells into the S-phase.
 25. The method of any one of claims 20 to 24wherein the candidate compounds are selected from a syntheticcombinatorial library.
 26. The use of a fragment of p16 proteinincluding the amino acid motifs FLD, corresponding to amino acidresidues 90 to 92 of full length p16 protein, and/or LVVL, correspondingto amino acid residues 94 to 97 of full length p16 protein in the designof an organic compound which is modelled to resemble the threedimensional structure of said amino acid motifs, the organic compoundhaving the properties of binding to cyclin dependent kinase and/orinhibiting pRb phosphorylation.